Osteoporosis is a debilitating disease that affects 75 million people worldwide with an estimated $48 billion in healthcare costs. A major determinant of bone mass is the mechanical loading to which the skeleton is subjected during daily activity, which stimulates adaptive modeling. Osteocytes, cells embedded in the bone matrix, are thought to be responsible for sensing and coordinating adaptive responses in the skeleton. In response to mechanical loading, osteocytes at sites experiencing high mechanical stimulus appear to be the initial perceivers of applied load and to translate this force into early biochemical signals that lead to bone formation. However, little is currently known about how these cells individually and collectively sense and integrate strains perceived over the entire bone in order to initiate site-specific adaptive modeling responses. Wnt signaling mediated by sclerostin and b-catenin, molecules that negatively and positively influence the Wnt/b-catenin pathway, clearly plays an important role in bone formation. Our preliminary data suggests that b-catenin is rapidly activated in osteocytes following mechanical loading. Our data also suggests that the load signal is propagated to adjacent cells, to sites where sclerostin protein expression is down regulated. Based on this, our overall hypothesis is that b-catenin signaling is controlled by magnitude of mechanical stimulus in a subset of osteocytes that activate the pathway followed by its subsequent propagation to adjacent regions when Sost becomes down regulated. In this application, we propose to use b-catenin signaling and Sost expression as readouts to determine how a driver of bone formation (b-catenin) and its negative regulator (Sost, an osteocyte specific molecule) are controlled by mechanical loading in osteocytes within a three dimensional mechanically responsive bone model. This in vivo model integrates strain or fluid flow shear stress magnitudes as the mechanical stimulus (a cause) with reporter activity or gene expression (a mechanism) with bone formation, a biological response (an effect). The following specific aims are proposed, Aim 1). Determine the temporal and spatial activation of the b-catenin pathway in response to anabolic load, Aim 2). Determine the temporal and spatial down regulation of Sost expression in response to anabolic load, and Aim 3). Determine the effects of increasing or decreasing b-catenin signaling on Sost/sclerostin expression and mechanical stimulus thresholds required to induce bone formation. We propose that increasing the basal level of b-catenin activity decreases the threshold of mechanical stimulus required to elicit bone formation in response to load and vice versa. Changes in b-catenin activity and Sost/sclerostin expression will be correlated with strain or fluid flow shear stress magnitudes in 3 dimensions. Thinking and visualizing bone responses to mechanical load in 3 dimensions instead of 2 dimensions will provide novel insights into how bone responds to load and will contribute to our understanding of the fundamental relationship between strain and bone cell responses. PUBLIC HEALTH RELEVANCE. Osteoporosis, a disease of low bone mass, is a debilitating disease that afflicts 75 million people worldwide with an estimated $48 billion in healthcare costs. A major determinant of bone mass is the mechanical loading to which the skeleton is subjected during daily activity, which stimulates adaptive modeling. Osteocytes, cells embedded in the bone matrix, are thought to be responsible for sensing and coordinating adaptive responses in the skeleton. In this application, we evaluate the dosage of a pharmacological agent (lithium chloride) that can synergize with mechanical loading to activate a powerful pathway (the Wnt/b-catenin signaling) in osteocytes such that it leads to bone formation occurring at sites of peak mechanical stresses. It is noted that failure to enhance bone formation at these sites can result in bone fractures. We also evaluate the activation of molecules that are involved in the pathway and show the activation of these molecules and bone formation in relationship to sites of peak mechanical stimuli in three dimensions. Therefore, this research is important in building an insightful foundation for regulation of bone mass by mechanical loading and in evaluating molecular pathways to reduce fracture risk. [unreadable] [unreadable] [unreadable]